Bio-Plex Pro RBM Apoptosis Assays

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Acute Phase Apoptosis Cancer Cardiovascular Disease Cytokines Chemokines, Growth Factors Diabetes Gene Expression Genotyping Immunoglobulin Isotyping MicroRNA Expression Bio-Plex Pro RBM Apoptosis

quality controls High-Performance Multiplex Immunoassays for Apoptosis Research The Bio-Plex Pro RBM apoptosis assays, developed in partnership with Myriad RBM, comprise a highly relevant set of

Myriad s close collaboration with SAIC-Frederick, Inc (now Leidos Biomedical Research, Inc.

1 Acute Phase Apoptosis Cancer Cardiovascular Disease Cytokines Chemokines, Growth Factors Diabetes Gene Expression Genotyping Immunoglobulin Isotyping MicroRNA Expression Bio-Plex Pro RBM Apoptosis Assays MAGNETIC SEPARATION ENABLED,, Lamin B, Smac,, /Bcl-2 Dimer, Bcl-xL, Bim, Mcl-1, Active Caspase-3, Bcl-xL/ Dimer, Mcl-1/ Dimer, and Survivin Magnetic workflow All-in-one kit format 2-level quality controls High-Performance Multiplex Immunoassays for Apoptosis Research The Bio-Plex Pro RBM apoptosis assays, developed in partnership with Myriad RBM, comprise a highly relevant set of intracellular proteins involved in the commitment, onset, and induction of apoptosis by the intrinsic pathway (Figure 1). Myriad s close collaboration with SAIC-Frederick, Inc (now Leidos Biomedical Research, Inc., which operates labs at NCI) was instrumental in the selection and clinical validation of markers found in these panels (Table 1). The assays are built on magnetic beads to enable robust quantification of multiple proteins in cell and tissue lysates. Assays are offered as premixed all-in-one kits for research involving: n Cancer cell biology n Mechanism of action of cancer drugs n Neurodegenerative diseases (such as Alzheimer s or Parkinson s) n Stroke n Heart failure n Viral infection (such as hepatitis) Assay features n Optimized for high precision and lot-to-lot reproducibility of sample measurements n Magnetic beads for simplified plate processing n 2-level quality controls with lot-specific ranges n Assay quick guide to get you started right away n Compatible with Bio-Plex 2, Bio-Plex 3D, and Bio-Plex MAGPIX systems Assay Performance Definitions The following parameters are indicative of assay performance, as shown in Table 2. Assay working range the range of concentrations within which the assay is precise and accurate. Boundaries of the assay working range are defined by the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) Precision the coefficient of variation (%CV) at concentrations within the assay working range Accuracy (recovery) percentage of the observed concentration relative to the expected concentration of a known amount of analyte within the assay working range Sensitivity (limit of detection, LOD) the concentration of analyte for which the fluorescence intensity signal is two standard deviations above the background signal

2 EXTRINSIC PATHWAY FasL INTRINSIC PATHWAY (Chemotherapy, irradiation, DNA-damaging agents) Survival factors: cytokines and growth factors Fas/ CD95 McI-1 FADD Caspase-8, 1 BID Bim PUMA MITOCHONDRIA BcI-2 BcI-xL PKA Erk1/2 PI3K/Akt p Caspase-3 Mcl-1 Smac/Diablo Cytosolic sequestration Cytochrome C Survivin (IAPs) Caspase-3, 7, 8 Caspase-9 APAF-1 APOPTOSOME FORMATION ICAD Vimentin Lamin B Actin Cytosol NUCLEUS APOPTOSIS Fig. 1. Apoptosis is induced by at least two distinct signaling pathways, the extrinsic and intrinsic pathway. The extrinsic pathway is triggered by signaling through death receptors such as Fas, followed by downstream activation of caspase-8 and caspase-3. The intrinsic pathway is triggered by cytotoxic stress, which leads to translocation of Bcl-2 family proteins, and, to the mitochondrial membrane. Oligomerization of and causes release of cytochrome C into the cytosol, which promotes apoptosome formation, caspase activation, degradation of nuclear lamin B, and cell death. proteins included in the panels ( ); anti-apoptotic proteins included in the panels ( ); proteins involved in apoptosis but not included in the panels ( ). 213 Bio-Rad Laboratories, Inc. Bulletin 6474

3 Table 1. Bio-Plex Pro RBM apoptosis analytes. Short Name Lamin B, intact and 45 kd Smac /Bcl-2 dimer Bcl-xL Bim Mcl-1 Active caspase-3 Bcl-xL/ dimer Mcl-1/ dimer Survivin Description Bcl-homologous antagonist/killer is a pro-apoptotic member of the Bcl-2 family. In healthy cells, it is integrated in the mitochondrial outer membrane. Upon apoptotic stimuli, forms oligomer channels in the mitochondrial membrane for cytochrome C release. This activity is regulated by forming a complex with anti-apoptotic Mcl-1 and Bcl-xL. Bcl-2 associated protein X is a pro-apoptotic member of the Bcl-2 family. In healthy cells, it is found as a monomer in the cytosol, but upon apoptotic stimuli, it translocates to the mitochondrial outer membrane, and forms large oligomeric complexes. There it interacts with pore proteins to enable cytochrome C release into the cytosol, and initiate the caspase activation pathway for apoptosis. may cycle to the mitochondrial membrane and dimerize with Bcl-xL. Nuclear lamins are proteins of intermediate filament type, located at the outer rim of the nucleus. They consist of two types of polypeptides, lamin A and lamin B. Lamin B consists of B1 and B2 subtypes. Lamins mechanically stabilize the cell nucleus and also play a role in DNA replication and chromatin organization. Lamin B is cleaved by caspase-3 and caspase-6 during the early phases of apoptosis (up to 9 min) before DNA fragmentation. Detection of cytoplasmic dissociated lamin B indicates cell apoptosis. Second mitochondria-derived activator of caspase is a dimeric mitochondrial protein synthesized in the cytoplasm as a 239 amino acid precursor protein, with 55 amino acids at the N-terminus serving as a mitochondrial-targeting sequence. Under apoptotic stimuli, it is proteolytically cleaved to a 23 kd active form and released into the cytosol together with cytochrome C, where it reverses IAP inhibition of caspase-9, allowing caspase-9 to activate the caspase cascade. Bcl-2-associated death promoter is a pro-apoptotic, BH3-only binding domain member of the Bcl-2 family. BH3-only proteins connect apoptotic death signals to the activation of and, which control mitochondrial membrane disruption and apoptosis. Phosphorylated (p) is typically bound to the cytosolic protein , and is thus sequestered away from the mitochondria. Dephosphorylation results in the release of cytosolic (free), which binds to and inhibits the pro-survival activity of Bcl-2 family proteins at the mitochondrial membrane. B-cell lymphoma-2 is an anti-apoptotic protein that resides on the outer mitochondrial membrane. When bound to as a heterodimer, it inhibits permeability of the mitochondrial membrane, preventing release of cytochrome C. B-cell lymphoma-extra large is an anti-apoptotic member of the Bcl-2 family found in the outer mitochondrial membrane. Heterodimerization with pro-apoptotic proteins (especially ) inhibits apoptosis by preventing release of cytochrome C. Bcl-2-interacting mediator of cell death is a pro-apoptotic protein belonging to the BH3-only group of the Bcl-2 family. Bim binds and antagonizes pro-survival members of the Bcl-2 family such as Mcl-1, Bcl-xL, and Bcl-2. Three prominent isoforms are generated by alternative splicing: Bim-S, Bim-L, and Bim-EL. Each isoform has the ability to induce apoptosis. Induced myeloid leukemia cell differentiation protein-1 is an anti-apoptotic member of the Bcl-2 family. Heterodimerization with pro-apoptotic proteins (especially ) inhibits apoptosis. While Mcl-1 may not be as potent a protector against apoptosis as Bcl-2, it does appear to be the main anti-apoptotic protein in some cell types including neutrophils. Cysteinyl aspartyl protease-3 belongs to the peptidase C14A enzyme family and is known to play an important role in the apoptotic cascade. The active enzyme is formed by cleavage of the inactive 32 kd pro-enzyme into the p17 and p12 subunits. Two of each subunit noncovalently heterodimerize giving the final enzyme two catalytic sites. Active caspase-3 cleaves and activates other caspases and is a primary regulator of apoptotic-associated proteolysis. Bcl-xL heterodimerizes with at the mitochondrial outer membrane and inhibits permeability of the mitochondrial membrane, preventing release of cytochrome C. During apoptosis BH3-only proteins, such as Bim and, will bind to Bcl-xL and cause to be released. Upon release, will oligomerize creating pores in the mitochondrial membrane. Mcl-1 heterodimerzes with and at the mitochondrial outer membrane to prevent their activation, thus inhibiting cytochrome C release from the mitochondria. Survivin is a 16 kd member of the Inhibitor of Apoptosis (IAP) family which also plays a role in chromosome segregation and cytokinesis. The anti-apoptotic function of survivin comes from its ability to inhibit the activation of caspases-3 and -7. Survivin is found to be upregulated in various tumors. Apoptotic Action Early indicator of apoptosis Apoptotic Cell membrane Moves to the mitochondrial membrane Cytosol Cytosol Cytosol (free) membrane; decreased with apoptosis membrane Cytosol or mitochodrial membrane if dimerized Mainly mitochondrial membrane, some in cytosol Cytosol (high levels) membrane; decreased with apoptosis Mitochondria; decreased with apoptosis Cytosol/nucleus 213 Bio-Rad Laboratories, Inc. Bulletin 6474

4 Table 2. Representative assay performance. Assay Working Range, ng/ml Assay Sensitivity, ng/ml Assay Precision Target Bead Region LLOQ ULOQ LOD Intra-assay %CV Inter-assay %CV Panel % 14% % 19% Lamin B, intact and 45 kd % 14% Smac % 19% Panel % 9% /Bcl-2 dimer % 9% Bcl-xL % 7% Bim % 8% Mcl % 6% Panel 3 Active caspase % 8% Bcl-xL/ dimer % 14% Mcl-1/ dimer % 13% Survivin % 8% The LLOQ, ULOQ, LOD, and inter-assay precision %CV are mean data determined from three independent multiplex assays. Intra-assay %CV is the mean of eight standard points run in triplicate within one representative assay. LLOQ and ULOQ are defined as the boundary standard curve points that meet precision and accuracy specifications of 3% intra-assay CV and 8 12% recovery. Data were generated using the magnetic workflow with the Bio-Plex Pro II wash station. Accuracy and Sensitivity Exceptional quality of the Bio-Plex Pro RBM apoptosis assays ensures high accuracy and sensitivity. The overall accuracy of the assays is provided by the standard curves generated in Bio-Plex Manager software. Standard curves were obtained for, active caspase-3, and Smac (Figure 2). Sensitivity was analyzed by comparing total and Bcl-xL assays with that of western blotting (Figure 3). Lower protein concentration levels of each apoptotic marker were detectable with the Bio-Plex Pro RBM apoptosis assays than with western blotting methods. Fluorescence intensity, Fi 1, 1, 1 1 1, ULOQ (2.216) S1(1) ULOQ (44.947) S2 (11) ULOQ ( ) S2 (1) 1, S3 (98) 1, S5 (13) S6 (97) S8 (89) S7 (18) S4 (98) S3 (12) LLOQ (.81) 1 1 Active Caspase-3 S6 (99) S7 (14) S8 (96) S5 (98) S4 (13) S1 (1) LLOQ (.21) 1, 1 1 S8 (82) S7 (112) S6 (99) Smac S5 (98) S4 (1) S3 (12) S1 (98) S1 (11) LLOQ (.62) Concentration, ng/ml Concentration, ng/ml Concentration, ng/ml Fig. 2. Standard curves with assay controls and cell lysates. Standard points were prepared by serially diluting a reconstituted standard threefold to generate an eight-point standard curve. Standard points with % recovery ( ); controls (s); samples (s). Data were generated in Bio-Plex Data Pro software. Total Total Bcl-xL 4, 4, 3, 3, MFI 2, MFI 2, 1, 1, IP Ab: (Lx capture), Westen Ab: (Lx detection) IP Ab: Bcl-xL (Lx capture), Westen Ab: Bcl-xL (Lx detection) Fig. 3. Sensitivity of Bio-Plex Pro RBM apoptosis assay compared to western blotting. A lysate purified from the nuclear + mitochondrial fraction of an untreated PC3 xenograft cell line was serially diluted and then measured using the Bio-Plex Pro RBM apoptosis kit and western blotting methods. The Bio-Plex assays demonstrated superior sensitivity down to the single digit µg/ml protein loading. 213 Bio-Rad Laboratories, Inc. Bulletin 6474

5 Expression Pattern of Apoptotic Markers Expression levels of pro-apoptotic and anti-apoptotic markers were established in healthy and cancer colon biopsies as well as tissue culture samples treated with the cancer drugs GSP, ABT, and HA14-1 (Figures 4 and 5). 8 Smac.5 Bcl-xl 11 Active Caspase Mcl-1 1 Mcl-1/ Fig. 4. Detection of apoptosis biomarkers in colon cancer tissue. A representative tissue was processed to compare the levels of apoptosis biomarkers in two sample fractions (nuclear + mitochondrial and cytosolic) relative to a non-matching normal colon tissue. Nuclear + mitochondrial (n); cytosolic (n). 28 Lamin B HCT116-Lysate 2 HCT116-Lysate Bcl-xL Bim 11 Active caspase-3 Bcl-xL/ HCT116-Lysate Gossypol dosage, µm ABT dosage, µm ABT dosage, µm 135 Lamin B Active caspase-3 MCF-7-Lysate 15 MCF-7-Lysate Bcl-xL 13 Bcl-xl-, ng/ml Bcl-xL/ Active caspase-3 MCF-7-Lysate ABT dosage, µm HA14-1 dosage, µm HA14-1 dosage, µm Fig. 5. Drug induced apoptosis. HCT-116 (ATCC CCL-247 ) and MCF7 (ATCC HTB-22 ) cells were treated for 3 hours with increasing concentrations of gossypol (, 5, 2, 4, 6, and 8 µm), ABT-263 or HA14-1 (, 2, 4, 6, 8 and 1 µm). Whole cell lysates were analyzed with the multiplex assays. 213 Bio-Rad Laboratories, Inc. Bulletin 6474

6 Ordering Information Catalog # 171-WAR1CK 171-WAR2CK 171-WAR3CK Description Bio-Plex Pro RBM Apoptosis Panel 1, 1 x 96-well all-in-one kit that includes premixed magnetic capture beads and detection antibodies, standards, 2-level controls, standard diluent, buffers (blocking, lysate dilution (LDB), cytosolic extraction (CEB), 1x assay), 1x streptavidin-pe, flat bottom plate, plate seals, and instructions, for the detection of the following analytes in cell and tissue lysates:,, lamin B, and Smac Bio-Plex Pro RBM Apoptosis Panel 2, 1 x 96-well all-in-one kit that includes premixed magnetic capture beads and detection antibodies, standards, 2-level controls, standard diluent, buffers (blocking, lysate dilution (LDB), cytosolic extraction (CEB), 1x assay), 1x streptavidin-pe, flat bottom plate, plate seals, and instructions, for the detection of the following analytes in cell and tissue lysates:, /Bcl-2 dimer, Bcl-xL, Bim, and Mcl-1 Bio-Plex Pro RBM Apoptosis Panel 3, 1 x 96-well all-in-one kit that includes premixed magnetic capture beads and detection antibodies, standards, 2-level controls, standard diluent, buffers (blocking, lysate dilution (LDB), cytosolic extraction (CEB), 1x assay), 1x streptavidin-pe, flat bottom plate, plate seals, and instructions, for the detection of the following analytes in cell and tissue lysates: Active caspase-3, Bcl-xL/ dimer, Mcl-1/ dimer, and survivin Wash Stations and Accessories Bio-Plex Pro Wash Station, microplate wash station for magnetic bead based assays, includes magnetic plate carrier, waste bottle, 2 liquid bottles Bio-Plex Handheld Magnetic Washer, includes magnetic washer and adjustment hex tools for use in manual wash steps for all Bio-Plex magnetic assays Bio-Plex Pro Flat Bottom Plates, pkg of 4, 96-well plates, for use with Bio-Plex Pro wash stations when using magnetic bead based assays Software Bio-Plex Data Pro Software with Bio-Plex Manager Software, Bio-Plex Data Pro software (5 seats), for multi-experiment analysis and advanced data visualization, and Bio-Plex Manager software (5 seats), for instrument data evaluation and optimization. CDs and security HASP key included Bio-Plex Data Pro Software, (5 seats), for multi experiment analysis and advanced data visualization 171-STND1 Bio-Plex Manager Software, includes 1 user desktop license, for analysis of Bio-Plex data and generation of protocols, does not operate the instrument The Bio-Plex suspension array system includes fluorescently labeled microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation. HASP is a trademark of Aladdin Knowledge Systems, Ltd. Myriad RBM is a trademark of Myriad RBM, Inc. Bio-Plex Pro RBM kits are manufactured by Myriad RBM. Bio-Rad Laboratories, Inc. Life Science Group Web site USA Australia Austria Belgium Brazil Canada China Czech Republic Denmark Finland France Germany Greece Hong Kong Hungary India Israel Italy Japan Korea Mexico The Netherlands New Zealand Norway Poland Portugal Russia Singapore South Africa Spain Sweden Switzerland Taiwan Thailand United Kingdom Bulletin 6474 Rev A US/EG Sig 1212

Quick Guide QUICK GUIDE. Activity 1: Determine the Reaction Rate in the Presence or Absence of an Enzyme

Quick Guide QUICK GUIDE. Activity 1: Determine the Reaction Rate in the Presence or Absence of an Enzyme Activity 1: Determine the Reaction Rate in the Presence or Absence of an 1. Find your 15 ml conical tubes labeled Stop Solution, 1.5 mm Substrate, and Buffer. Write your initials on each tube. 2. Label